1 INTRODUCTION
Traditionally, most freshwater mollusks inventories were based on shells
records (ex. Duncan, 2008). The durability of shells may be an advantage
for species detection but may lead to potential biases. Although shell
surveys are less time consuming, they often overestimate population
sizes, given that there is always more dead than alive specimens. More
importantly, they can also be misleading regarding species occurrences.
We have many examples of species being extirpated from a site where
shells can still be found for many years. Furthermore, the traditional
classification of freshwater mollusks is based on plastic shell traits
that make identification difficult without taxonomical expertise. This
turns surveys based on shells only a difficult task for some species and
a source of numerous species misidentifications. This is especially true
for large and conspicuous species such as the Unionoid bivalves. For
smaller shells, like the fingernail clams, another important bias in
rivers, although poorly documented, is shell drifting, where shells can
be collected far away from their source population.
Therefore, most freshwater mollusks atlases based on these classical
surveys are likely biased. In these publications, misidentified
specimens, ancient data, and shell-only data are pooled together
generally leading to enlarged species distributions and improved
freshwater mollusks conservation status. Our results based on a
large-scale environmental DNA (eDNA) investigation shine a light on
these biases and contribute to more reliable species occurrences and
therefore to more accurate conservation status assessments.
DNA barcodes provide more reliable species identifications than shell
morphology (Prié, Puilandre, & Bouchet, 2012; Prié & Puillandre, 2014;
Riccardi et al., 2019). DNA analyses are sometimes used to confirm
protected species determination, e.g. for the Thick-shelled River MusselUnio crassus Philipsson, 1788, a protected species in European
countries, which is sometimes difficult to identify but may have
important economic impacts, if found in an area planned for project
development. However, several difficulties hamper the use of DNA
barcodes for species identification, among which lab availability,
price, permits to sample protected species, as sampling tissue for DNA
can be hazardous for mollusks, and the need to find and collect live
specimens, which can be a difficult and time-consuming task. Freshwater
mollusks are hard to survey, sometimes difficult to detect, especially
for small species or species that spend most of their life buried.
Freshwater bivalves surveys often involve scuba diving, which means
having specialized biologists, costly equipment, and also increased
associated risks (e.g. navigation, drifting objects, deadwood, fishing
lines, etc…) which have to be accounted for, especially in a
professional context.
The development of non-invasive genetic monitoring methods with the
extraction of environmental DNA (eDNA) from indirect sources, such as
fur, scats, or soil and water samples, brought important advances in the
remote detection and monitoring of species that are rare and/or
difficult to capture or sample (Taberlet, Coissac, Hajibabaei, &
Rieseberg, 2012). eDNA metabarcoding analyses are therefore a promising
way to overcome the difficulties regarding freshwater mollusks’ surveys
and sampling. They rely on a barcoding approach, which secures
determinations. Samples can be easily and safely collected in the field
(contrarily to scuba diving or river prospecting), do not require
particular skills, and provide data about the extant living population,
as eDNA cannot be detected for long in freshwater (Dejean et al., 2011;
Pont et al., 2018).
eDNA analyses have been developed for about 10 years to survey
freshwater biodiversity (eg. amongst others Ficeola, Miaud, Pompanon &
Taberlet, 2008; Dejean et al., 2011; Taberlet et al. 2012; Darling &
Mahon, 2012… see Thomsen & Willerslev, 2015 for a review). Most
studies are still based on single-species detection and generally have a
limited sampling plan (e.g., Goldberg, Sepulveda, Ray, Baumgardt, &
Waits, 2013; Evans et al., 2015; Stoeckle, Huehn, & Geist, 2016; De
Ventura, Kopp, Seppälä, & Jokela, 2017; Klymus, Marshall, & Stepien,
2017). Here, we present the first extensive dataset using standardized
eDNA metabarcoding protocols for freshwater bivalves surveys, at the
scale of whole France, with about 350 sampling sites. The present
results reveal some inaccuracies in our previous knowledge of species
distributions. As the extent and changes in distribution are one of the
main parameters used for species conservation status assessments, this
change of paradigm may have a strong impact on future conservation
policies.